Intercellular communication is essential to coordinate the behaviour of individual cells during organismal development. The preimplantation mammalian embryo is a paradigm of tissue self-organization and regulative development; however, the cellular basis of these regulative abilities has not been established. Here we use a quantitative image analysis pipeline to undertake a high-resolution, single-cell level analysis of lineage specification in the inner cell mass (ICM) of the mouse blastocyst. We show that a consistent ratio of epiblast and primitive endoderm lineages is achieved through incremental allocation of cells from a common progenitor pool, and that the lineage composition of the ICM is conserved regardless of its size. Furthermore, timed modulation of the FGF-MAPK pathway shows that individual progenitors commit to either fate asynchronously during blastocyst development. These data indicate that such incremental lineage allocation provides the basis for a tissue size control mechanism that ensures the generation of lineages of appropriate size.

f5: EPI cells trigger PrE specification but not vice versa.(a) Diagram of the treatment regimes embryos were subject to. (b) Scatter plots for GATA6 and NANOG levels (expressed as logarithm) in individual ICM cells, for each treatment condition after 30 h in culture. Contour lines have been overlaid as density estimators. Control: KSOM or KSOM+1 μg ml−1 of heparin; FGF4: KSOM+1 μg ml−1 rhFGF4+1 μg ml−1 of heparin; MEKi: KSOM+1 μM PD0325901. Cell identity (colour coded) assigned using the same clusters as in Fig. 1d. Number of embryos (N) and cells analysed are indicated in each plot. (c) Representative immunofluorescence images of embryos analysed in b. NANOG (EPI) and GATA6 (PrE) shown in grayscale in ICM magnifications and colour coded in the merged panel. In merged image, CDX2 marks the TE lineage and ‘c' indicates the total number of cells of the embryo shown. All images are 5 μm Z projections. (d) Scatter plots for GATA6 and NANOG levels (as logarithm) in individual ICM cells, after release from FGF4/MEKi and culture in control medium for another 18 h. Contour lines have been overlaid as density estimators. Cell identity (colour coded) was assigned using the same clusters used as in Fig. 2b. Number of embryos (N) and cells analysed are indicated in each plot. (e) Representative immunofluorescence images of embryos analysed in d. (f) Average ICM composition at the end of the culture period for embryos treated in each of the conditions indicated. (g) Representative immunofluorescence images of an embryo treated for 30 h with FGF4 and cultured in MEKi for further 18 h, showing both EPI and PrE layers. (h) Scatter plot for GATA6 and NANOG levels (as logarithm) in individual ICM cells for embryos cultured as in g. Contour lines have been overlaid as density estimators. Cell identity was assigned as in d. Colour coding is indicated. DN, double negative (GATA6−, NANOG−); DP, double positive (GATA6+, NANOG+); EPI: epiblast (NANOG+); PRE, primitive endoderm (GATA6+). For a description of the criteria used to correct fluorescence levels and to determine cell identity, see Methods. Scale bar, 20 μm.

Mentions:
To experimentally test this model, we prevented specification of EPI cells by exposing 8-cell stage embryos to FGF4 for 30 h (50–64-cell stage; Fig. 5a). At this stage, all ICM cells displayed GATA6 expression, but had completely lost, or exhibited highly reduced levels of, NANOG (Fig. 5b,c)—unlike culture for 24 h only (Supplementary Fig. 7a–c,d). When embryos were released from FGF4 stimulation and cultured for a further 18 h in control medium, the large majority displayed only GATA6+ PrE cells (Fig. 5d–f and Supplementary Fig. 7e), suggesting that, indeed, PrE cells cannot specify EPI fate among their neighbours. Alternatively, FGF4 may remain bound to heparan sulfate proteoglycans57 and continue to stimulate the receptors. Embryos transferred from FGF4 to MEKi-containing medium for 18 h contained both EPI and PrE cells (Fig. 5g,h), suggesting that residual extracellular FGF4 may continue to stimulate the RTK–MAPK axis in progenitor cells or else that basal MEK activity is sufficient to prevent NANOG upregulation in these cells.

f5: EPI cells trigger PrE specification but not vice versa.(a) Diagram of the treatment regimes embryos were subject to. (b) Scatter plots for GATA6 and NANOG levels (expressed as logarithm) in individual ICM cells, for each treatment condition after 30 h in culture. Contour lines have been overlaid as density estimators. Control: KSOM or KSOM+1 μg ml−1 of heparin; FGF4: KSOM+1 μg ml−1 rhFGF4+1 μg ml−1 of heparin; MEKi: KSOM+1 μM PD0325901. Cell identity (colour coded) assigned using the same clusters as in Fig. 1d. Number of embryos (N) and cells analysed are indicated in each plot. (c) Representative immunofluorescence images of embryos analysed in b. NANOG (EPI) and GATA6 (PrE) shown in grayscale in ICM magnifications and colour coded in the merged panel. In merged image, CDX2 marks the TE lineage and ‘c' indicates the total number of cells of the embryo shown. All images are 5 μm Z projections. (d) Scatter plots for GATA6 and NANOG levels (as logarithm) in individual ICM cells, after release from FGF4/MEKi and culture in control medium for another 18 h. Contour lines have been overlaid as density estimators. Cell identity (colour coded) was assigned using the same clusters used as in Fig. 2b. Number of embryos (N) and cells analysed are indicated in each plot. (e) Representative immunofluorescence images of embryos analysed in d. (f) Average ICM composition at the end of the culture period for embryos treated in each of the conditions indicated. (g) Representative immunofluorescence images of an embryo treated for 30 h with FGF4 and cultured in MEKi for further 18 h, showing both EPI and PrE layers. (h) Scatter plot for GATA6 and NANOG levels (as logarithm) in individual ICM cells for embryos cultured as in g. Contour lines have been overlaid as density estimators. Cell identity was assigned as in d. Colour coding is indicated. DN, double negative (GATA6−, NANOG−); DP, double positive (GATA6+, NANOG+); EPI: epiblast (NANOG+); PRE, primitive endoderm (GATA6+). For a description of the criteria used to correct fluorescence levels and to determine cell identity, see Methods. Scale bar, 20 μm.

Mentions:
To experimentally test this model, we prevented specification of EPI cells by exposing 8-cell stage embryos to FGF4 for 30 h (50–64-cell stage; Fig. 5a). At this stage, all ICM cells displayed GATA6 expression, but had completely lost, or exhibited highly reduced levels of, NANOG (Fig. 5b,c)—unlike culture for 24 h only (Supplementary Fig. 7a–c,d). When embryos were released from FGF4 stimulation and cultured for a further 18 h in control medium, the large majority displayed only GATA6+ PrE cells (Fig. 5d–f and Supplementary Fig. 7e), suggesting that, indeed, PrE cells cannot specify EPI fate among their neighbours. Alternatively, FGF4 may remain bound to heparan sulfate proteoglycans57 and continue to stimulate the receptors. Embryos transferred from FGF4 to MEKi-containing medium for 18 h contained both EPI and PrE cells (Fig. 5g,h), suggesting that residual extracellular FGF4 may continue to stimulate the RTK–MAPK axis in progenitor cells or else that basal MEK activity is sufficient to prevent NANOG upregulation in these cells.

Intercellular communication is essential to coordinate the behaviour of individual cells during organismal development. The preimplantation mammalian embryo is a paradigm of tissue self-organization and regulative development; however, the cellular basis of these regulative abilities has not been established. Here we use a quantitative image analysis pipeline to undertake a high-resolution, single-cell level analysis of lineage specification in the inner cell mass (ICM) of the mouse blastocyst. We show that a consistent ratio of epiblast and primitive endoderm lineages is achieved through incremental allocation of cells from a common progenitor pool, and that the lineage composition of the ICM is conserved regardless of its size. Furthermore, timed modulation of the FGF-MAPK pathway shows that individual progenitors commit to either fate asynchronously during blastocyst development. These data indicate that such incremental lineage allocation provides the basis for a tissue size control mechanism that ensures the generation of lineages of appropriate size.